Background: Relapse and refractory disease remain major challenges in acute myeloid leukemia (AML), with venetoclax resistance significantly limiting treatment efficacy. Nuclear factor erythroid 2-related factor 2 (NRF2), a key transcriptional regulator of oxidative stress, is implicated in chemoresistance, but its role in venetoclax resistance remains unclear. This study investigates the NRF2-DDIT4 pathway as a novel mechanism of venetoclax resistance in AML.

Methods:

  • Clinical and bioinformatics analysis: NRF2 and DDIT4 expression was assessed in AML patient samples (remission vs. relapse/refractory) via RT-qPCR and Western blot. Public datasets (GEO, GTRD, KnockTF) were analyzed to identify NRF2-regulated genes linked to venetoclax resistance.

  • In vitro models: Venetoclax-resistant AML cell lines (THP1, MV4-11) were generated. NRF2/DDIT4 were overexpressed (lentivirus) or knocked down (shRNA), followed by apoptosis assays (flow cytometry).

  • Transcriptional regulation: ChIP-seq and dual-luciferase assays validated NRF2 binding to the DDIT4 promoter.

  • In vivo validation: AML xenograft models (MV4-11R) with DDIT4 knockdown were treated with venetoclax to assess tumor growth and survival.

  • Proteomics/metabolomics: Differentially expressed proteins (e.g., BIM) and metabolites (glutathione pathway) were analyzed in DDIT4-knockdown cells.

Results:

  • NRF2 and DDIT4 were significantly upregulated in relapsed/refractory AML and correlated with poor prognosis.

  • NRF2 overexpression reduced venetoclax sensitivity, while NRF2/DDIT4 knockdown restored sensitivity.

  • ChIP confirmed NRF2 binding to the DDIT4 promoter, and luciferase assays demonstrated NRF2-dependent DDIT4 transactivation.

  • DDIT4 knockdown in NRF2-overexpressing cells reversed resistance.

  • In xenografts, DDIT4 silencing enhanced venetoclax efficacy and prolonged survival.

  • Proteomics identified BIM downregulation in resistant cells, while metabolomics revealed glutathione metabolism dysregulation.

Conclusion: The NRF2-DDIT4 axis drives venetoclax resistance in AML via transcriptional activation of DDIT4, leading to BIM suppression and altered glutathione metabolism. Targeting this pathway may overcome resistance and improve outcomes in refractory AML.

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2025
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